Optimization of ultrahigh-speed multiplex PCR for forensic analysis

Abstract

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl, and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization

“…When dealing with nucleic acids, which are also conveyed from EVs and may work as carrier for mutations with a role in carcinogenesis, a convenient strategy is typically to exploit amplification methods for generating multiple copies of a target nucleic acid, either through standard PCR cycles or alternative approaches, such as isothermal amplification [ 207 , 212 ] and subsequently analyse the sequence looking for specific genes or point mutations. However, off-chip nucleic acid-based detection relies on complex analytical procedures and requires a relatively long time with multiple steps, expensive instruments and reagents, and trained personnel.…”

The development of miniSTRs as a method for high‐speed direct PCR

“…For a fast presumptive DNA analysis method that can be used when time is limited, the developed rapid direct two-step PCR method should be combined with high-speed microfluidic separation and detection methods. Previous studies have shown the use of microchips for separation and detection of DNA fragments [22,23,[41][42][43]. For example, Anazawa et al developed a multi-channel microchip electrophoresis system with five-color and eight-channel fluorescence detection.…”

“…Since 1990 direct PCR has been used in molecular biology [13][14][15]. Over the past 20 years, there has been a great deal of research performed focusing on the use of rapid direct PCR in the forensic field [4,12,[16][17][18][19][20][21][22][23]. Commercial direct PCR multiplex STR kits have been developed and validated for analyzing reference database samples [16][17][18][19], and crime samples [21].…”

“…The analysis time for PCR in these studies has been reduced to 60 min or less [11,21]. Gibson-Daw et al [22] have demonstrated an amplification of 7-locus multiplex (MP7) in 6.5 min using extracted DNA. Aboud et al [23] showed that it is possible to generate an MP7 DNA profile in under 25 min when using rapid direct PCR coupled to a microfluidic device using DNA from a punch taken from FTA paper containing saliva stains.…”

Optimisation of rapid STR analysis using a standard DNA forensic pipeline

“…In addition to improvements made by STR kit manufacturers, published studies looking at reducing the time taken to generate a DNA profile using standard laboratory equipment have focused on use of direct PCR for touch and trace DNA [5,6], use of enhanced DNA polymerase enzymes [5,7,8,9,10], modified PCR mixes [9,11], and use of more efficient thermal cycler instrumentation [10,12]. In addition, research has also been carried out in the field of microfluidic chip systems to separate and detect fragments in place of traditional capillary electrophoresis platforms [5,13].…”